Administration of tailored feedstock to increase nitro-containing amphenicol antibiotic susceptibility

ABSTRACT

A method for increasing susceptibility of microorganisms to antibiotics includes providing a microorganism; administering an antibiotic including a nitro-containing amphenicol compound to the microorganism; and administering any of an uronic, aldonic, ulosonic, and aldaric feedstock to the microorganism. The feedstock is adapted to promote cell metabolism, and inhibit antibiotic inactivation pathways in the microorganism causing increased sensitivity of the microorganism to the nitro-containing amphenicol.

This application is related to U.S. patent application Ser. No.15/939,329 filed Mar. 29, 2018, the disclosure of which is incorporatedby reference in its entirety. The '329 application discloses method forincreasing susceptibility of microorganisms to antibiotics comprising anitroimidazole compound. Nitroimidazoles are a class of chemicalcompounds with active imidazole ring and nitro group at 2′- or5′-positions. They work mainly on anaerobic bacteria or parasites, andcan also be used in tumor treatments.

GOVERNMENT INTEREST

The embodiments herein may be manufactured, used, and/or licensed by orfor the United States Government without the payment of royaltiesthereon.

BACKGROUND Technical Field

The embodiments herein generally relate to a method for increasingsusceptibility of microorganisms to antibiotics, and more particularly,to nitro-containing amphenicol antibiotic compounds.

Description of the Related Art

There are many other families and classes of antibiotics. Amphenicolsare broad spectrum antibiotics that can treat aerobic/anaerobic grampositives and gram negatives including and not limited to: Rickettsia,Chlamyophila, Mycoplasma, Salmonella, Enteracter, Klebsiella,Escherichia, Pseudomonas aeruginosa, Proteus, H. influenzae,Streptococcus pneumoniae and Neisseria meningitidis. They have aphenylpropanoid structure and function by blocking the enzyme peptidyltransferase on the 50S ribosome subunit of bacteria.

While nitroimidazoles are similar to a prodrug, in that, in order forthem to be active and act as an antibiotic, they need to be reduced inthe cell to an active intermediate. The active intermediate is a freeradical and tends to cause DNA breaks and other affects. On the otherhand, amphenicols are active as administered. Both nitroimidazole activeintermediate and amphenicols are inactivated once they are furtherreduced.

Over use of these antibiotics has caused a significant increase inantibiotic resistance, resulting in longer hospital stays and increaseddrug costs. Additionally, use of antibiotics kills the natural gutflora, which can result in intestinal inflammation and possibly fataldiarrhea in children.

Amphenicol antibiotics are typically used as a last resort for multidrugresistant infections due to serious negative side effects. Use ofamphenicol has been attributed to bone marrow suppression, aplasticanemia and increased susceptibility to Clostridium difficile infections.The annual costs of C. difficile treatment is estimated to be $6.3billion dollars a year and multidrug resistant infections isapproximately $2 billion a year, not to mention the cost of treatingeach infection individually.

Like nitroimidazole antibiotic compounds discussed in the aforementioned'329 application, there are no current methods or additives to increasesusceptibility of cells to amphenicol antibiotic compounds unless anadditional antibiotic is administered either. Therefore, there is a needto develop a more convenient approach to increase susceptibility ofmicroorganisms to amphenicol antibiotics in fighting infections, therebyreducing side effects associated with antibiotic treatment.

Amphenicol compounds exist including those having nitro- (—NO₂) andsulfur oxide- (—SO₂) functional groups. This application is specific tothe amphenicol compounds containing a nitro- (—NO₂) functional group.

SUMMARY

In view of the foregoing, an embodiment herein provides a method forincreasing susceptibility of microorganisms to nitro-containingamphenicol antibiotic, the method comprising providing a microorganism;administering an antibiotic comprising a nitro-containing amphenicolcompound to the microorganism; and administering any of an uronic,aldonic, ulosonic, and aldaric feedstock to the microorganism, whereinthe feedstock is adapted to promote cell metabolism and inhibitantibiotic inactivation pathways in the microorganism causing increasedsensitivity of the microorganism to the nitro-containing amphenicol. Thenitro-containing amphenicol compound may comprise chloramphenicol orazidamfenicol. The feedstock may comprise a sugar acid or a combinationof sugar acids comprising any of an uronic, aldonic, ulosonic, andaldaric acid.

The method may comprise metabolizing galacturonate from the galacturonicacid by producing adenosine triphosphate (ATP) and reduced ferredoxin.The feedstock may be adapted to decrease production of NADH and NADPH inthe microorganism. The method may comprise administering the feedstockas a supplement for oral antibiotics. The feedstock may comprise anaqueous solution or a solid form. The method may comprisedecontaminating the microorganism. The method may comprise administeringthe feedstock by intravenous injection, subcutaneous injection, orintraperitoneal injection.

BRIEF DESCRIPTION OF THE DRAWINGS

The embodiments herein will be better understood from the followingdetailed description with reference to the drawings, in which:

FIG. 1 shows the chemical structures of two known nitro-containingamphenicol antibiotic compounds: chloramphenicol and azidamfenicol.

FIG. 2 is a schematic illustrating C. acetobutylicum metabolism andinteractions with a nitro-containing amphenicol compound; and

FIG. 3 is a graph illustrating C. acetobutylicum sensitivity tochloramphenicol after 24 hours; and

FIG. 4 is a flow diagram illustrating a method of administering anitro-containing amphenicol compound to a microorganism.

DETAILED DESCRIPTION

The embodiments herein and the various features and advantageous detailsthereof are explained more fully with reference to the non-limitingembodiments that are illustrated in the accompanying drawings anddetailed in the following description. Descriptions of well-knowncomponents and processing techniques are omitted so as to notunnecessarily obscure the embodiments herein. The examples used hereinare intended merely to facilitate an understanding of ways in which theembodiments herein may be practiced and to further enable those of skillin the art to practice the embodiments herein. Accordingly, the examplesshould not be construed as limiting the scope of the embodiments herein.

Embodiments of the present invention provide methods to increasemicroorganisms' susceptibility to nitro-containing amphenicol antibioticcompounds. As mentioned above, amphenicols have a phenylpropanoidstructure and function by blocking the enzyme peptidyl transferase onthe 50S ribosome subunit of bacteria. They can treat aerobic/anaerobicgram positives and gram negatives including and not limited to:Rickettsia, Chlamyophila, Mycoplasma, Salmonella, Enteracter,Klebsiella, Escherichia, Pseudomonas aeruginosa, Proteus, H. influenzae,Streptococcus pneumoniae and Neisseria meningitidis.

FIG. 1 shows the chemical structure for two nitro-containing amphenicolantibiotic compounds: chloramphenicol and azidamfenicol. Chloramphenicolis the common form of nitro-containing amphenicols having anantimicrobial activity and a broad spectrum of action on bacteriaincluding Staphylococcus aureus, Streptococcus pneumoniae, andEscherichia coli. It is commonly produced synthetically. For treatment,it is typically administered topically, orally, or injected.Azidamfenicol has a similar profile to chloramphenicol. It is typicallyused topically, such as in eye drops and ointments.

Antibiotics are more effective in cells with active metabolism.Metabolism of the nitro-containing amphenicol compounds inmicroorganisms may be altered to decrease eukaryotic cytotoxicintermediates (which have been found to result in bone marrowsuppression and aplastic anemia) through the selected administration offeedstock as an additive or supplement to control metabolic flux of NADHfor decrease metabolic flux through pathways for nitro-containingamphenicol inactivation. For example, intracellular NADH can bedecreased through the administration of oxidized feedstock such asgalacturonic acid to Clositridium sp. By lowering the availability ofNADH in a microorganism, there is a decrease in inactivation of thenitro-containing amphenicol which increases its susceptibility in thatmicroorganism. Additionally, this allows bacterial infections to beselectively targeted through the administration of specific feedstocksmetabolized only by that species, leaving normal flora alone. Thismethod allows for treatment of infections without the harmful sideeffects.

The methods for increasing microorganisms' susceptibility tonitro-containing amphenicol antibiotics comprise administering theuronic, aldonic, ulosonic, and/or aldaric substrates or feedstocks. Moreparticularly, the embodiments herein provide for administering effectiveamount of nitro-containing amphenicol and uronic, aldonic, ulosonic,and/or aldaric substrates or feedstocks to microorganisms.

Particular embodiments use substrates; e.g., uronic, aldonic, ulosonic,and/or aldaric acids or salts that promote cell metabolism but alsoinhibit or decrease flux through pathways required for antibioticinactivation and/or repair of antibiotic damage. There may be still someflux, but not as much as with other feedstocks, decreasing the amount ofNADH. In particular embodiments, intracellular NADH may be decreasedthrough the administration of oxidized substrates such as galacturonicacid or glucuronic acid to C. acetobutylicum. Referring now to thedrawings, and more particularly to FIGS. 2 through 4, where similarreference characters denote corresponding features consistentlythroughout the figures, there are shown preferred embodiments.

“Antibiotics” as used herein refers to drug used in the treatment andprevention of bacterial infections, or any substance used againstmicrobes.

“Microorganism” as used herein refers to unicellular or mutlicellularorganisms, includes bacteria, archaea, protists, protozaons, oreukaryotes; e.g., human, animal, and plant cells.

“Anaerobic organism” as used herein refers to any organism that may notor must not require oxygen for growth, organism may be unicellular ormulticellular organism.

“Feedstock” and “Substrate” may be used interchangeably and is definedas material comprising carbohydrate or sugar (e.g. glucose, gluconate,and galacturonate).

“Effective amount” is used herein to denote a quantity or concentrationof that antibiotics and/or substrate is known to be effective to achievethe desired and known result of the antibiotics and/or substrate. Theactual amount contained in the molecular complex or composition, likelywill vary since some of the antibiotics and/or substrate composition maynot completely penetrate the microorganism together. Using theguidelines provided herein, those skilled in the art are capable ofdetermining the acceptable amount of antibiotics and substrate describedherein, and to use the requisite amount. For example, a suitable dosageadjustment may be made by the attending physician or veterinariandepending upon the age, sex, weight and general health of the subject.Such a composition may be administered parenterally, optionallyintramuscularly or subcutaneously. However, the composition may also beformulated to be administered by any other suitable route, includingorally or topically.

In another embodiment, the nitro-containing amphenicol compound of thecomposition may include, but are not limited to, compounds such aschloramphenicol and azidamfenicol.

A common carbohydrate such as glucose a type of aldose, is oxidized atcarbon one position from aldehyde to carboxyl group, the product iscalled aldonic acid, or more specifically gluconic acid. The aldonicacid usually has multiple hydroxyl groups. The general chemical formulais HOOC—(CHOH)_(n)—CH₂OH. Oxidation of the terminal hydroxyl groupoccurs instead of the terminal aldehyde and yields a uronic acid, whileoxidation of both terminal ends yields an aldaric acid. Aldonic acid mayexist as stereoisomers as D, L, and DL or R, S and RS forms. HenceD-glucose is oxidized to D-gluconic acid and D-gluconolactone.

In one embodiment, any of the uronic, aldonic, ulosonic, and aldaricfeedstock may be an aqueous solution or solid form. Thus, for example itmay be in tablet, coated tablet, delayed or sustained release coatedtablet, capsule, suppository, pessary, gel, emulsion, syrup, dispersion,suspension, emulsion, powder, cream, paste, etc.

In another embodiment, any of the uronic, aldonic, ulosonic, and aldaricfeedstock may be administered as a supplement for oral antibiotics, suchas an antibiotic chaser in a shake or drink form.

In one embodiment, any of the uronic, aldonic, ulosonic, and aldaricfeedstock may be administered with two or more different therapeuticcompounds; e.g., with two different antibiotics. Two differentantibiotics with substrates may be administered either in the sameformulation or in a separate formulation, either concomitantly orsequentially.

Antibiotics against anaerobes microorganisms include penicillin G,amphenicol, imipenem, ampicillin-sulbactam, clindamycin, cefoxitin,piperacillin, ceftizoxime, cefoperazone, erythromycin, moxalactam,cefotetan, cefipime or a combination of antibiotics.

In one embodiment, nitro-containing amphenicol; e.g., chloramphenicol,is effective for the treatment of anaerobic infections, such asintra-abdominal infections, gynecologic infections, septicemia,endocarditis, bone and joint infections, central nervous systeminfections, respiratory tract infections, skin and skin-structureinfections, and oral and dental infections.

In another embodiment, nitro-containing amphenicol and any of theuronic, aldonic, ulosonic, and aldaric feedstock may be used with otherantibiotics for treatment of mixed aerobic and anaerobic infection, orin combination with other antibacterial agents that are appropriate forthe treatment of the aerobic infection, or other anaerobic infections.

The composition of the embodiments herein may be administered to anypart, organ, interstice or cavity of a human or non-human body that issubject to an infection or radiation. For example, the composition maybe administered by, but not limited to, oral and non-oral preparations(e.g., intramuscular, subcutaneous, transdermal, visceral, IV(intravenous), IP (intraperitoneal), intraarticular, placement in theear, ICV (intracerebralventricular), intraarterial, intrathecal,intracapsular, intraorbital, injectable, pulmonary, nasal, rectal, anduterine-transmucosal preparations).

In some embodiments, a process of decontaminating the surface occurs byapplying the feedstock or substrate with antibiotics to a surface thatis contaminated with one or more microbes. Any delivery mechanism fordecontaminating a surface may be used including spraying, immersing, orother contact mechanism.

FIG. 2 illustrates C. acetobutylicum metabolism and interactions with anitro-containing amphenicol compound with respect to three feedstocks:glucose, gluconate, and galacturonate. Conversion of galacturonate, andglucose to acetyl-CoA produces similar amounts of ATP and reducedferredoxin to help drive metabolism and nitro-containing amphenicolinactivation. Further, conversion of galacturonate and glucose toacetyl-CoA produces different amounts of NADH.

More particularly, FIG. 2 shows how three feedstocks (i.e., glucose,gluconate, and galacturonate) are assimilated into central metabolism ofthe C. acetobutylicum. The feedstocks may in the form of substrates asconventionally used in in vitro experiments, for instance. All threefeedstocks produce two moles of pyruvate per mole of the feedstockcompound and the pyruvate is converted to acetyl-CoA and CO₂ by pyruvateferredoxin oxidoreductase (PFOR). PFOR, the hydrogenase, and reducedferredoxin have all been shown to inactivate nitro-containingamphenicols. On a molar basis, all three feedstocks produce equivalentamounts of reduced ferredoxin and metabolize equivalent levels ofpyruvate via PFOR. The feedstocks differ in the amount of NADH that isproduced as shown in Table 1 below. Per mole of glucose the cellsproduce 2 moles of NADH upstream of pyruvate while during growth ongalacturonate there is no net NADH production. The lack of NADHproduction reduces the capacity of the organism to inactivate thenitro-containing amphenicols via nitroreductases or other relatedenzymes.

TABLE 1 NADH/NADPH Production NADH/NADPH produced upstream of SubstratePFOR glucose 2 gluconate 1 galacturonate 0

Experimental evidence suggests microorganism (e.g., cells) should bemore sensitive to nitro-containing amphenicols when grown ongalacturonate because there is no net NADH/NADPH produced. NADH andNADPH are required to reduce amphenicols to their inactive form. Glucoseand nitro-containing amphenicols metabolism produces net NADH/NADPHupstream of PFOR which can be used to inactivate those amphenicols vianitroreductases and the cells should therefore be less susceptible tothose amphenicols on these substrates when compared to galacturonate.

The feeding strategy provided by the embodiments herein and described inFIG. 2 may be utilized for a broad range of anaerobic organisms due toevolutionary conservation of the pathway for galacturonate metabolismand the need to reduce the substrate for entry into glycolysis.Depending on the target organism other feeding strategies, such as useof different aldonic and uronic acid carbohydrates, may be used to meetthe criteria of providing ATP and reduced ferredoxin while minimizingthe production of NADH/NADPH. Accordingly, by decreasing the breakdownof the intermediates by the cells, the sensitivity increases.

The two main sources of reduced electron carriers in C. acetobutylicumare NADH from lower glycolysis and reduced ferredoxin from PFOR.Electron carriers are reoxidized by the hydrogenase, which couplesferredoxin oxidation with proton reduction, and by reductive conversionof acetyl-CoA to butyrate. Cells gain one ATP per acetyl-CoA byconversion to acetate, as opposed to 0.5 ATP per acetyl-CoA generatedduring reduction to butyrate. It is therefore more favorable from thestandpoint of ATP yield to use the hydrogenase to reoxidize electroncarriers. Electrons may be shuffled between NADH and ferredoxin by theNADH-ferredoxin oxidoreductase or butyryl-CoA dehydrogenase, thus it ispossible for reducing equivalents formed in lower glycolysis to beoxidized indirectly via the hydrogenase.

Unlike nitroimidazole antibiotic compounds which require activating bythe cells, nitro-containing amphenicols antibiotic compounds do not.That is because amphenicols are a class of antibiotics with aphenylpropanoid structure which functions by blocking the enzymepeptidyl transferase on the 50S ribosome subunit of bacteria. The drugcan be inactivated by a reduction step. On the other hand,nitroimidazoles have nonspecific action and thus needs to be activatedonce inside the bacteria cell through a reduction to an intermediatethat has a free radical. The free radical on the chemical causes DNAdamage and other damage. The intermediate is inactivated through furtherreduction.

Inactivation of nitro-containing amphenicols can occur through severaldifferent mechanisms. The bacteria reduce nitro groups (—NO₂) to anamine (—NH₂). This process requires nitroreductases or other redoxenzymes to reduce the nitro groups while oxidizing intracellularelectron carriers such as NADH, NADPH, thioredoxin, flavodoxin,rubredoxin, etc. The nitro-containing amphenicol compound leads toinhibition of protein expression which is repaired by the reversiblebinding of the antibiotic to the ribosome. Thus, when the microorganism(e.g. cells) are fed nitro-containing amphenicol compounds, the resultis decreased production of NADH and NADPH, which in turn causes areversal in ferredoxin oxidation/reduction cycle via the hydrogenasetowards oxidized ferredoxin. As a result, the cells are metabolicallyactive, but have diminished capacity to reduce nitro-containingamphenicol to their inactive form and a diminished capacity to expressproteins. The toxic intermediates formed from the reduction andinactivation of the antibiotic have been known to possibly cause severeside effects such as bone marrow suppression and aplastic anemia.

The combined effect is that bacterial cells have increased sensitivityto chloramphenicol and in turn diminishes the formation of eukaryoticcytotoxic intermediates of the antibiotic (resulting in bone marrowsuppression and aplastic anemia).

In another embodiment, administering oxidized feedstock to C.acetobutylicum, antibiotic susceptibility increases at least one and ahalf fold or greater compared to administering glucose as a feedstock.

In another embodiment, the different components of the feedstocks may bepackaged together with antibiotics or in separate containers. Ifappropriate, and mixed immediately before use, such packaging of thecomponents separately may permit long-term storage without losing theactive component's function. Sterilization may be preceded or followedby packing into containers. If desired, the composition of theembodiments herein may contain pharmaceutically acceptable additives,such as dissolving aids, buffering components, stabilizers, and thelike. The antibiotics and/or substrates may be supplied in containers ofany sort such at the life of the different components are preserved andare not adsorbed or altered by the materials of the container. Forexample, sealed glass ampules may contain lyophilized substrates andvariants, derivatives and structural equivalents thereof, or buffersthat have been packaged under a neutral, non-reacting gas, such asnitrogen. Other containers include test tubes, vials, flasks, bottles,syringes, or the like. Containers may have a sterile access port, suchas a bottle having a stopper that may be pierced by a hypodermicinjection needle. Other containers may have two compartments that areseparated by a readily removable membrane that upon removal permits thecomponents to be mixed. Removable membranes may be glass, plastic,rubber, etc.

Suitable pharmaceutically acceptable carriers facilitate administrationof the antibiotic and substrate or feedstocks are physiologically inertand/or nonharmful. Carriers may be selected by one skilled in the art.Exemplary carriers include sterile water or saline, calcium phosphate,gelatin, dextran, agar, pectin, peanut oil, olive oil, sesame oil, andwater. Additionally, the carrier or diluent may include a time delaymaterial, such as glycerol monostearate or glycerol distearate alone orwith a wax. In addition, slow release polymer formulations may be used.

The substrates or feedstock with or without antibiotic provided by theembodiments herein may additionally contain stabilizers such asthimerosal (ethyl(2-mercaptobenzoate-S)mercury sodium salt) (availablefrom Sigma Chemical Company, St. Louis, Mo.), for example, orphysiologically acceptable preservatives.

The composition provided by the embodiments herein may also containconventional pharmaceutical ingredients, such as preservatives, orchemical stabilizers. Suitable ingredients operable herein include, forexample, casamino acids, gelatin, phenol red, N—Z amine, monopotassiumdiphosphate, lactalbumin hydrolysate, and dried milk.

The nitro-containing amphenicol may be formulated into a composition ina free base, neutral or salt form. Pharmaceutically acceptable saltsinclude the salts formed with a free carboxyl group or amine groupderived from inorganic bases such as for example, sodium, potassium,ammonium, calcium or ferric hydroxides; or such organic bases asisopropylamine, trimethylamine, histidine or procaine. The antibioticsand/or substrates may be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms. It will be appreciated that endotoxin contaminationshould be kept minimally at a safe level.

Bacterial culture conditions and strains have been previously published.All strains and cultures were maintained or grown in in an atmosphere of5.0% H₂, 5.0% CO₂, and 90.0% N₂ . Clostridium acetobutylicum strain ATCC824 was obtained from ATCC and cultured using company protocol at 37° C.into Clostridial growth medium or CGM containing 0.75 g KH₂PO₄, 0.75 gK₂HPO₄, 1.0 g NaCl, 0.017 g MnSO₄.5H₂O, 0.70 g MgSO₄.7H₂O, 0.01 gFeSO₄.7H₂O, 2.0 g 1-asparagine, 5.0 g yeast extract, 2.0 g (NH₄)₂SO₄,and 0.5% final concentration of desired carbohydrate—D-glucose,D-galacturonic acid, D-gluconic acid—at pH 6.5. Active cultures frominitial growth stock were added to potato glucose medium to bemaintained and stored as a spore solution. Potato glucose medium or PGMcontains per liter of H₂O—150 g grated fresh potato, 10 g D-glucose, 0.5g (NH₄)SO₄, and 3 g CaCO₃. The medium was boiled for 1 hour and strainedthrough gauze before sterilization and use for culture. Spore solutionwas activated for culturing through a heat shock at 80° C. for 9minutes. Shocked spore solution was added to CGM containing the feedstock of choice and grown to late log of 0.8 at optical density of 600nm (OD₆₀₀) at 37° C. Theses cultures were then used to performantibiotic sensitivity experiments.

The protocol for antibiotic sensitivity test was performed as follows inanaerobic conditions: 200 μL of CGM medium containing either 0.5%D-galacturonate or D-glucose was aliquoted into a sterile Costar®96-well polystyrene flat bottom plate in triplicate per test condition.Chloroamphenicol was dissolved in ethanol at different concentrations.Each chloroamphenicol concentration was added to each well in triplicateand considered a test condition. Test conditions included addition of 1μL of ethanol, 1 of sterile water, 1 μL 0.1 mg mL⁻¹, 1 μL 0.25 mg mL⁻¹,1 μL 0.5 mg mL⁻¹, 1 μL 1 mg mL⁻¹, 1 μL 2.5 mg mL⁻¹, 1 μL 5 mg mL⁻¹, 1 μL10 mg mL⁻¹, 1 μL 20 mg mL⁻¹, and 1 μL 10 mg mL⁻¹. 10 μL of OD₆₀₀ 0.8Clostridium acetobutylicum culture grown in CGM containing 0.5%D-galacturonate was aliquoted into each test condition containingD-galacturonic acid. 10 of OD₆₀₀ 0.8 Clostridium acetobutylicum culturegrown in CGM containing 0.5% D-glucose was aliquoted into each testcondition containing D-glucose. The final concentrations of amphenicolafter addition of culture are as follows 0.0 μg mL⁻¹, 0.0 μg mL⁻¹, 0.47μg mL⁻¹, 1.18 μg mL⁻¹, 2.37 μg mL⁻¹, 4.74 μg mL⁻¹, 11.85 μg mL⁻¹, 23.7μg mL⁻¹, 47.4 μg mL⁻¹, 94.8 μg mL⁻¹, 142.0 μg mL⁻¹. The 96-well platewas covered and incubated for 20 hours at 37° C. The optical density ofthe cultures in the wells were measured at 600 nm via a commerciallyavailable plate reader.

As illustrated in FIG. 3, administering oxidized feedstock to C.acetobutylicum, antibiotic (chloraamphenicol) susceptibility increasesduring growth on galacturonate when compared to growth on glucose.Galacturonate and gluconate produce similar amounts of ATP and reducedferredoxin when compared to glucose to help drive metabolism. Metabolismof gluconate and galacturonate results in less production of NADH/NADPHwhen compared to growth on glucose which impairs chloramphenicolinactivation and repair of oxidative damage. In vitro experiments showthat administering oxidized feed stock to anaerobic bacteria, C.acetobutylicum, antibiotic susceptibility increases 10 fold compared toadministering glucose as a feed stock. The MIC for galacturonic acid forfinal concentration of chloramphenicol is 2.4 ng/μL and for glucose is23.7 ng/μL after 24 h of growth. This experiment has been performseveral times and was based off of the proposed model.

FIG. 4 is a flow diagram illustrating a method 100 for increasingsusceptibility of microorganisms to nitro-containing amphenicolantibiotics, according to an embodiment herein. The method 100 comprisesproviding (101) a microorganism; administering (102) an antibioticcomprising a nitro-containing amphenicol compound to the microorganism;and administering (103) any of an uronic, aldonic, ulosonic, and aldaricfeedstock to the microorganism, wherein the feedstock is adapted topromote cell metabolism and inhibit antibiotic inactivation pathways inthe microorganism causing increased sensitivity of the microorganism tothe nitro-containing amphenicol. In one example, the administeringprocesses (102) and (103) may be sequential. In another example, theadministering processes (102) and (103) may be simultaneous. Thenitro-containing amphenicol compound may comprise chloramphenicol orazidamfenicol. The feedstock may comprise a sugar acid or a combinationof sugar acids comprising any of an uronic, aldonic, ulosonic, andaldaric acid.

The method may comprise metabolizing gluconate from the glucuronic acidby producing adenosine triphosphate (ATP) and reduced ferredoxin. Thefeedstock may be adapted to decrease production of NADH and NADPH in themicroorganism. The method may comprise administering the feedstock as asupplement for oral antibiotics. The feedstock may comprise an aqueoussolution or a solid form. The method may comprise decontaminating themicroorganism. The method may comprise administering the feedstock byintravenous injection, subcutaneous injection, or intraperitonealinjection.

The embodiments herein decrease the concentration of nitro-containingamphenicol antibiotic required for treatment through the use ofnaturally occurring feedstocks. The embodiments herein increase thesusceptibility of the targeted cells to nitro-containing amphenicols,without requiring an additional antibiotic to be administered. Byutilizing specific naturally occurring feedstocks as compared tosynthetic chemicals or additional antibiotics, the embodiments hereindecrease the chance of possible side effects to the patient and decreasethe cost of production.

Furthermore, the embodiments herein decrease the concentration of thenitro-containing amphenicol antibiotic required for treatment bytargeting specific cells for susceptibility as compared to naturalflora. Targeting occurs because not all bacteria can grow on the samefeedstock and there for can be tailored for bacteria of interest. Thismay decrease antibiotic production costs and possible patient sideeffects (e.g., diarrhea, intestinal inflammation, liver damage, etc.).Additionally, by controlling the cells NADH production, the embodimentsherein may decrease antibiotic resistance through the inhibition ofantibiotic breakdown which requires energy input.

The embodiments herein may be used in various capacities, including as atreatment for numerous types of infections, control of microbiomes tosupport health and improve performance, as well as for decontaminationof biohazardous environments. Additionally, the tailored feedstock usedby the embodiments herein may be used as an additive to suppositories,topical creams and ointments, eye drops, injections, and liquid oralantibiotics emulsions. The tailored feedstock may be administered as asupplement for oral antibiotics such as an “antibiotic chaser” in ashake form or drink form, in various examples. Additionally, thetailored feedstock may be made as a shake or additive to a diet plan formeals prepared for the patient to support antibiotic susceptibility.

Furthermore, the embodiments herein may be used as a feedstock to makethe bacterial infection more susceptible to nitro-containing amphenicolsthrough the control of the metabolic flux of NADH/NADPH by acting as anactive ingredient to reduce antibiotic inactivation. The method providedby the embodiments herein offers a unique solution in that it may beused as a feedstock and active ingredient and not an additive forhydration/moisturizing and/or as part of a salt for the antibiotic as aPPI to help deal with the pH in the patient's stomach.

The embodiments herein increase the nitro-containing amphenicolantibiotic sensitivity by feeding cells (e.g., microorganisms,multicellular organisms) substrates that promote metabolism but inhibitpathways required for antibiotic inactivation and/or repair ofantibiotic damage.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the embodiments herein that others may, byapplying current knowledge, readily modify and/or adapt for variousapplications such specific embodiments without departing from thegeneric concept, and, therefore, such adaptations and modificationsshould and are intended to be comprehended within the meaning and rangeof equivalents of the disclosed embodiments. It is to be understood thatthe phraseology or terminology employed herein is for the purpose ofdescription and not of limitation. Therefore, while the embodimentsherein have been described in terms of preferred embodiments, thoseskilled in the art will recognize that the embodiments herein may bepracticed with modification within the spirit and scope of the appendedclaims.

What is claimed is:
 1. A method for increasing susceptibility ofmicroorganisms to antibiotics, the method comprising: providing amicroorganism; administering a single type of an antibiotic comprising anitro-containing amphenicol compound to the microorganism; andadministering any of an uronic, aldonic, ulosonic, and aldaric feedstockto the microorganism, wherein the feedstock is adapted to promote cellmetabolism and inhibit antibiotic inactivation pathways in themicroorganism causing increased sensitivity of the microorganism to thenitro-containing amphenicol.
 2. The method of claim 1, wherein thenitro-containing amphenicol compound comprises chloramphenicol orazidamfenicol.
 3. The method of claim 1, wherein the feedstock comprisesa sugar acid or a combination of sugar acids comprising any of uronic,aldonic, ulosonic, and aldaric acid.
 4. The method of claim 3,comprising metabolizing galacturonate from galacturonic acid byproducing adenosine triphosphate (ATP) and reduced ferredoxin.
 5. Themethod of claim 1, wherein the feedstock is adapted to decreaseproduction of NADH and NADPH in the microorganism.
 6. The method ofclaim 1, comprising administering the feedstock as a supplement for oralantibiotics.
 7. The method of claim 1, wherein the feedstock comprisesan aqueous solution or a solid form.
 8. The method of claim 1,comprising decontaminating the microorganism.
 9. The method of claim 1,comprising administering the feedstock by intravenous injection,subcutaneous injection, or intraperitoneal injection.
 10. A method forincreasing susceptibility of microorganisms to antibiotics, the methodcomprising: administering an antibiotic comprising a nitro-containingamphenicol compound to a microorganism; and administering any of anuronic, aldonic, ulosonic, and aldaric feedstock to the microorganism,wherein the feedstock is adapted to promote cell metabolism and inhibitantibiotic inactivation pathways in the microorganism causing increasedsensitivity of the microorganism to the nitro-containing amphenicol.